Carboxylic true ionophores were previously demonstrated to have efficient antimalarial activity against the human parasite Plasmodium falciparum, with a 50% inhibitory concentration around nM and generally high selectivity as compared to their toxic effects against mammalian cell lines. The decreased molecular packing of the erythrocyte membrane outer leaflet after malarial infection could explain the preferential ionophore interaction with infected erythrocytes. Monolayer penetration experiments using different phospholipid films showed strong incorporation of true carboxylic ionophores, from classes 1 (nigericin) and 2 (lasalocid), up to a surface pressure close to film collapse. The interaction was slightly higher with PC (phosphatidylcholine) monolayers than with monolayers composed of cholesterol-containing total lipid extracts from either malaria-infected or normal erythrocytes, and the two latter induced identical interactions with 5-bromo lasalocid, Surface pressure-area isotherms for pure ionophores on water and surface tension of ionophore aqueous solutions clearly highlighted the surface-active characteristics of these ionophores and allowed determination of their molecular area in compact monolayers, The estimated ionophore concentration in the mixed interfacial layers indicates that higher amounts (threefold more) of ionophores might be integrated in infected erythrocyte membrane due to their impaired molecular packing as compared to normal erythrocytes. This infection-enhanced penetration efficiency does not appear directly related to the change in erythrocyte membrane lipid composition, but it could be the basis of ionophore selectivity for infected erythrocytes.